Connexin29 expression, immunocytochemistry and freeze-fracture replica immunogold labelling (FRIL) in sciatic nerve

Abstract

The recently discovered connexin29 (Cx29) was reported to be present in the central and peripheral nervous systems (CNS and PNS), and its mRNA was found in particular abundance in peripheral nerve. The expression and localization of Cx29 protein in sciatic nerve were investigated using an antibody against Cx29. The antibody recognized Cx29 in HeLa cells transfected with Cx29 cDNA, while nontransfected HeLa cells were devoid of Cx29. Immunoblotting of sciatic nerve homogenate revealed monomeric and possibly higher molecular weight forms of Cx29. These were distinguished from connexin32 (Cx32), which also is expressed in peripheral nerve. Double immunofluorescence labelling for Cx29 and Cx32 revealed only partial colocalization of the two connexins, with codistribution at intermittent, conical-shaped striations along nerve fibers. By freeze-fracture replica immunogold labelling (FRIL), Cx32 was found in gap junctions in the outermost layers of myelin, whereas Cx29-immunogold labelling was found only in the innermost layer of myelin in close association with hexagonally arranged intramembrane particle (IMP) 'rosettes' and gap junction-like clusters of IMPs. Although both Cx32 and Cx29 were detected in myelin of normal mice, only Cx29 was present in Schwann cell membranes in Cx32 knockout mice. The results confirm that Cx29 is a second connexin expressed in Schwann cells of sciatic nerve. In addition, Cx29 is present in distinctive IMP arrays in the inner most layer of myelin, adjacent to internodal axonal plasma membranes, where this connexin may have previously unrecognized functions.

Fig. 1

Western blots of Cx29 in sciatic nerve and Cx32 in liver. (A) Sciatic nerve homogenate probed with anti-Cx29 (lane 1) and, shown for comparison, liver homogenate probed with anti-Cx32 (antibody 73F, lane 2) and with anti-Cx29 (lane 3). Monomer and putative higher molecular weight forms of Cx29 are detected in nerve, and multiple forms of Cx32 are detected in liver. Cx29 migrates slightly slower than Cx32, and anti-Cx29 does not react with Cx32 in liver. (B) Sciatic nerve homogenate probed with anti-Cx29 (lane 1), and with anti-Cx29 after preadsorption with immunizing peptide (lane 2) showing elimination of all bands.

Fig. 2

(A) Western blots of Cx29 in HeLa cells transiently transfected with Cx29 cDNA. Lysates from two separate cultures of cells transfected with Cx29 expression vector show antibody detection of monomeric Cx29 (lanes 2 and 3) corresponding to that of Cx29 in homogenate of sciatic nerve (lane 1), while no detection is seen in control HeLa cells transfected with empty vector (lane 4). (B and C) Immunofluorescence of Cx29 in transfected HeLa cells showing robust immunolabelling in a small percentage of cells (B), and total absence of labelling in cells transfected with empty vector (C). Inset shows immunofluorescence within a pair of cells and around their periphery. Scale bar, 100 μm (B and C), 25 μm (inset).

Fig. 3

Immunofluorescence of Cx29 in sections of sciatic nerve from CD1 mice. (A) Low magnification showing distribution of labelling with anti-Cx29 antibody. (B and C) Magnifications showing labelling of either conical structures (B, arrows) or bands (B, arrowhead) orientated perpendicular to the long axis of fibers, and accumulation of staining at nodes of Ranvier (C, arrows). (D and E) Laser scanning confocal micrographs of the same field at different planes of focus showing collar of immunolabelling midway through a fibre (D, arrow) and closer to its surface (E, arrow). Note absence of labelling within the axon in D. (F) Confocal micrograph showing accumulation of labelling for Cx29 on each side (arrowheads) of a node of Ranvier (arrow). Scale bars, 100 μm (A); 25 μm (B and C); 5 μm (D–F).

Fig. 4

Double immunofluorescence labelling of Cx29 with Cx32 and MAG in mouse sciatic nerve. (A and B) Same field of a section showing labelling with anti-Cx29 (A) and labelling for Cx32 with monoclonal antibody 73F (B). (C and D) Same field of a section showing labelling with anti-Cx29 (C) and labelling for Cx32 with monoclonal antibody 7C7. (E and F) Same field of a section showing labelling with anti-Cx29 (E) and with anti-MAG (F). Corresponding arrows in (A and B), (C and D) and (E and F) indicate double-labelled structures. Scale bars, 50 μm.

Fig. 5

Specificity of immunofluorescence labelling of Cx29. (A) Field in a section of sciatic nerve processed with anti-Cx29 antibody, and (B) the same field in an adjacent section showing elimination of labelling after preadsorption of anti-Cx29 with immunizing peptide. The field shown is the apex of a hairpin loop in the section, resulting in curvature of fibers. (C and D) The same field in a section of liver processed for immunofluorescence with anti-Cx32 and anti-Cx29 antibody. Punctate labelling occurs around hepatocytes with anti-Cx32 7C7 (A) and no labelling is present with anti-Cx29 (B), indicating lack of cross reaction of the latter with Cx32. Scale bar, 50 μm.

Fig. 6

Double immunofluorescence for Cx29 and Cx32 in sciatic nerve of Cx32 knockout mice (strain C57BL/6). (A and B) The same field in a section from Cx32 knockout mouse processed with anti-Cx29 antibody (A) and monoclonal anti-Cx32 (B) antibody 73F. (C) Normal labelling for Cx32 in sciatic nerve of wild-type mouse strain C57BL/6. Absence of labelling in section devoid of Cx32 (B) indicates lack of cross-reaction of anti-Cx32 antibody with Cx29. Scale bars, 50 μm.

Fig. 7

FRIL images of gap junctions labelled for Cx32 in normal adult mouse sciatic nerve. (A) Gap junction between outer tongue and second layer of myelin (M) (boxed area). A multistranded tight junction (TJ) also links the outer tongue E-face with the P-face of second myelin layer. (B) Magnification of the boxed gap junction in A. The E-face of the junction is visualized, with connexins of the underlying hemiplaque labelled for Cx32 by four 5-nm gold beads (arrowhead) and two 6-nm gold beads (arrow). In this reverse (‘intaglio’) stereoscopic image, gold beads appear on top of the inverted replica for easier visualization. (C) P-face of the second outermost layer of myelin showing a gap junction composed of seven connexon IMPs labelled for Cx32 by a single 18-nm gold bead. Cytoplasm within the outer tongue of myelin is indicated by an asterisk. Scale bars, 0.1 μm.

Fig. 8

Stereoscopic images of P-face IMPs in the innermost myelin layer (M) of sciatic nerve after double-labelling for Cx32 and Cx29. (A and B) Normal sciatic nerve showing two IMP rosettes (boxed area in A, magnified in B and further magnified in B, inset). Each rosette is labelled for Cx29 (12-nm gold). (C) Sciatic nerve from Cx32 KO mouse. IMPs in rosettes and clusters are immunogold labelled for Cx29, with both 6- and 18-nm gold beads present beneath P-face IMPs. Labeling extends beneath the replicated, partially dislodged fragment. Magnified image in inset shows interlocking IMP rosettes in association with clusters of similar 8-nm IMPs labelled with four 6-nm and four 18-nm gold beads. No labelling for Cx32 (12-nm gold) was detected beneath rosettes or IMP clusters in innermost myelin layers of normal or Cx32 KO mice. Ax, Axon; M, cross-fractured myelin. Scale bars, 0.1 μm.

Fig. 9

IMP rosettes and clusters in innermost myelin layer in sciatic nerve of Cx32 KO mouse after double-labelling for Cx29 (6 and 18-nm gold) and Cx32 (12-nm gold; none present). Label for Cx29 is abundant beneath P-face IMPs in the innermost layer of myelin (A, inscribed boxes), but was absent in other layers of myelin (A, lower left) or in areas of knife scrape (K) (A, centre-right). (B) Magnification of lower box in A. The 6-nm gold beads (arrows) are more easily seen in areas where shadowed debris prevented platinum deposition. In the area not coated with platinum, IMPs are faintly delineated by the very thin, rotary-deposited carbon film. (C) Magnified upper box in A showing clustered IMPs labelled with six 18-nm and 24 6-nm gold beads (arrows). The 6-nm gold beads are more readily evident by first viewing the intaglio images (right two images), then the true stereoscopic perspective (left two images). Scale bars, 1 μm (A); 0.1 μm (B and C).